Comparison of three PET dopamine D2-like receptor ligands, [11C]raclopride, [11C]nemonapride and [11C]N-methylspiperone, in rats

We studied the tracer kinetics of three dopamine D2-like receptor ligands, [11C]raclopride ([11C]RAC), [11C]nemonapride ([11C]NEM) and [11C]N-methylspiperone ([11C]MSP), in anesthetized rats by tissue dissection, ex vivo ARG and PET in order to clarify their characteristics for PET imaging. The in vivo affinity of the three ligands for the striatum ([11C]MSP > [11C]NEM > [11C]RAC) obeyed the in vitro affinity for dopamine D2 receptors. The affinity of [11C]RAC and [11C]MSP for the cerebellum was very low, but the affinity of [11C]NEM for the cerebellum was compatible to that for the cortex and was not to be ignored. Also the affinity of [11C]MSP for the cortex was relatively high. [11C]RAC showed the highest selectivity. The striatal PET image with [11C]RAC was clearer than that with [11C]NEM or [11C]MSP, but the activity decreased much faster than that measured by tissue dissection because of the partial volume effect. The striatal activity with [11C]NEM remained high and that with [11C]MSP gradually increased. [11C]RAC and [11C]MSP, but not [11C]NEM, showed a high accumulation in the periorbital region.     

A preliminary PET evaluation of the new dopamine D2 receptor agonist [11C]MNPA in cynomolgus monkey

This study describes the preliminary positron emission tomography (PET) evaluation of a dopamine D(2)-like receptor agonist, (R)-2-(11)CH(3)O-N-n-propylnorapomorphine ([(11)C]MNPA), as a potential new radioligand for in vivo imaging of the high-affinity state of the dopamine D(2) receptor (D(2)R). MNPA is a selective D(2)-like receptor agonist with a high affinity (K(i)=0.17 nM). [(11)C]MNPA was successfully synthesized by direct O-methylation of (R)-2-hydroxy-NPA using [(11)C]methyl iodide and was evaluated in cynomolgus monkeys. This study included baseline PET experiments and a pretreatment study using unlabeled raclopride (1 mg/kg). High uptake of radioactivity was seen in regions known to contain high D(2)R, with a maximum striatum-to-cerebellum ratio of 2.23+/-0.21 at 78 min and a maximum thalamus-to-cerebellum ratio of 1.37+/-0.06 at 72 min. The pretreatment study demonstrated high specific binding to D(2)R by reducing the striatum-to-cerebellum ratio to 1.26 at 78 min. This preliminary study indicates that the dopamine agonist [(11)C]MNPA has potential as an agonist radioligand for the D(2)-like receptor and has potential for examination of the high-affinity state of the D(2)R in human subjects and patients with neuropsychiatric disorders.     

Quantitative ex vivo and in vitro receptor autoradiography using 11C-labeled ligands and an imaging plate: a study with a dopamine D2-like receptor ligand [11C]nemonapride.

Ex vivo and in vitro autoradiography (ARG) with radioluminography is a useful technique to characterize newly developed 11C-labeled positron emission tomography (PET) tracers and to apply them to biological and pharmacological studies. In this report, we have described a method of evaluating the radioactivity distribution quantitatively in ex vivo and in vitro ARG using imaging plates and a dopamine D2-like receptor ligand [11C]nemonapride as a model compound. The photo-stimulated luminescence (PSL) values of the rat brain section provided by the imaging plates showed an excellent linear relationship with the radioactivity in a wide range under constant slice-thickness, although the PSL values slightly decreased with increasing slice-thickness both in ex vivo and in vitro ARG. The injection dose of 11C-tracers for ex vivo ARG was also discussed. We found saturable binding sites of [11C]nemonapride in the cortex besides the striatum both ex vivo and in vitro.     

Test-retest reproducibility of dopamine D2/3 receptor binding in human brain measured by PET with [11C]MNPA and [11C]raclopride

Purpose

Dopamine D_2/3 receptors (D_2/3Rs) have two affinity states for endogenous dopamine, referred to as high-affinity state (D_2/3 ^HIGH), which has a high affinity for endogenous dopamine, and low-affinity state (D_2/3 ^LOW). The density of D_2/3 ^HIGH can be measured with (R)-2-¹¹CH₃O-N-n-propylnorapomorphine ([¹¹C]MNPA), while total density of D_2/3 ^HIGH and D_2/3 ^LOW (D_2/3Rs) can be measured with [¹¹C]raclopride using positron emission tomography (PET). Thus, the ratio of the binding potential (BP) of [¹¹C]MNPA to that of [¹¹C]raclopride ([¹¹C]MNPA/[¹¹C]raclopride) may reflect the proportion of the density of D_2/3 ^HIGH to that of D_2/3Rs. In the caudate and putamen, [¹¹C]MNPA/[¹¹C]raclopride reflects the proportion of the density of D₂ ^HIGH to that of D₂Rs. To evaluate the reliability of the PET paradigm with [¹¹C]MNPA and [¹¹C]raclopride, we investigated the test-retest reproducibility of non-displaceable BP (BP _ND) measured with [¹¹C]MNPA and of [¹¹C]MNPA/[¹¹C]raclopride in healthy humans.

Methods

Eleven healthy male volunteers underwent two sets of PET studies on separate days that each included [¹¹C]MNPA and [¹¹C]raclopride scans. BP _ND values in the caudate and putamen were calculated. Test-retest reproducibility of BP _ND of [¹¹C]MNPA and [¹¹C]MNPA/[¹¹C]raclopride was assessed by intra-subject variability (absolute variability) and test-retest reliability (intraclass correlation coefficient: ICC).

Results

The absolute variability of [¹¹C]MNPA BP _ND was 5.30?±?3.96 % and 12.3?±?7.95 % and the ICC values of [¹¹C]MNPA BP _ND were 0.72 and 0.82 in the caudate and putamen, respectively. The absolute variability of [¹¹C]MNPA/[¹¹C]raclopride was 6.11?±?3.68 % and 11.60?±?5.70 % and the ICC values of [¹¹C]MNPA/[¹¹C]raclopride were 0.79 and 0.80 in the caudate and putamen, respectively.

Conclusion

In the present preliminary study, the test-retest reproducibility of BP _ND of [¹¹C]MNPA and of [¹¹C]MNPA/[¹¹C]raclopride was reliable in the caudate and putamen.     

PET Imaging of the AT1 receptor with [11C]KR31173

AIM: The goal of this study was to investigate the binding characteristics of [(11)C]KR31173 and its applicability for PET studies of the AT(1) receptor (AT(1)R). METHODS: Ex vivo biodistribution and pharmacology were tested in mice. PET imaging was performed in mice, beagle dogs and a baboon. To assess nonspecific binding, PET imaging was performed both before and after pretreatment with a potent AT(1)R antagonist. In the baboon, PET imaging was also performed with the previously developed radioligand [(11)C]L-159,884 for comparison. RESULTS: Ex vivo biodistribution studies in mice showed specific binding rates of 80-90% in the adrenals, kidneys, lungs and heart. Specific binding was confirmed in mice using small animal PET. In dogs, renal cortex tissue concentration at 75-95 min postinjection (pi) was 63 nCi/ml per millicurie at a specific binding rate of 95%. In the baboon renal cortex, tissue activity at 55-75 min pi was 345 nCi/ml per millicurie. In the baboon the specific binding of [(11)C]KR31173 was higher (81%) than the specific binding of [(11)C]L-159,884 (34%). CONCLUSION: [(11)C]KR31173 shows accumulation and significant specific binding to the AT(1)R in the kidneys of mice, dogs and baboon. These findings suggest that this radioligand is suited for imaging the renal cortical AT(1)R in multiple species.     

Synthesis of [11C]SCH 23390 for dopamine D1 receptor studies

The optimal conditions for the synthesis of ¹¹C-labeled SCH 23390 by radio-methylation of its desmethyl precursor, SCH 24518, with [¹¹C]iodomethane are described. Isocratic reversed phase HPLC was used for the purification of [¹¹C]SCH 23390. The specific activity range in 30 runs was 10–235 Ci/mmol and average radiochemical yield was 72% based on [¹¹C]iodomethane. Mean synthesis time was 40–60 min from the end of bombardment. Preliminary animal studies indicate that [¹¹C]SCH 23390 would be useful in visualizing D1 receptors in a living brain by positron tomography.     

Preparation of 11C-labelled Raclopride, a new potent dopamine receptor antagonist: preliminary PET studies of cerebral dopamine receptors in the monkey

A new dopamine receptor antagonist, Raclopride (S-(-)-3,5-dichloro-N-[(1-ethyl-2-pyrrolidinyl)]methyl-2-hydroxy- 6-methoxybenzamide, FLA 870) (1), has been labelled using [11C]ethyl iodide for alkylation of the nitrogen of the pyrrolidine ring in the corresponding secondary amine (5). The synthesis of 5 and an efficient method for the preparation of [11C]ethyl iodide are described. The 11C-labelled FLA 870 (1) was purified by HPLC and then used in positron emission tomography to visualize the dopamine receptor-rich areas of the monkey brain. The images obtained show selective accumulation of FLA 870 in striatum and a 10-fold separation between the binding to caudate vs cerebellum.     

Positron emission tomography and ex vivo and in vitro autoradiography studies on dopamine D2-like receptor degeneration in the quinolinic acid-lesioned rat striatum: comparison of [11C]raclopride, [11C]nemonapride and [11C]N-methylspiperone

With [11C]raclopride,[11C]nemonapride and [11C]N-methylspiperone, degeneration of dopamine D2-like receptors in the unilaterally quinolinic acid-lesioned rats was evaluated by positron emission tomography (PET) and ex vivo and in vitro autoradiography. PET showed a decreased uptake of [11C]raclopride in the lesioned striatum, but an increased uptake of [11C]nemonapride and [11C]N-methylspiperone despite a decreased binding in vitro. Ex vivo autoradiography showed an increased accumulation of the three ligands in the cortical region overlying the injured striatum, probably enlarging PET signals. PET has the limited potential for evaluating the receptor degeneration in the present animal model.     

Development and evaluation of muscarinic cholinergic receptor ligands N-[11C]ethyl-4-piperidyl benzilate and N-[11C]propyl-4-piperidyl benzilate: a PET study in comparison with N-[11C]methyl-4-piperidyl benzilate in the conscious monkey brain

The muscarinic cholinergic receptor ligands N-[¹¹C]ethyl-4-piperidyl benzilate (4-EPB) and N-[¹¹C]propyl-4-piperidyl benzilate (4-PPB) were developed and evaluated in comparison with N-[¹¹C]methyl-4-piperidyl benzilate (4-MPB) in the conscious monkey brain using positron emission tomography (PET). Time-activity curves of [¹¹C]4-EPB, unlike [¹¹C]4-MPB, showed peaks within 91 min in regions rich in muscarinic receptors. [¹¹C]4-PPB showed no specific binding even in the regions rich in these receptors. These observation demonstrated that increases in [¹¹C]alkyl chain length could alter the kinetic properties of receptor ligands for PET.     

Imaging for metabotropic glutamate receptor subtype 1 in rat and monkey brains using PET with [18F]FITM

Purpose

In this study, we evaluate the utility of 4-[¹⁸F]fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide ([¹⁸F]FITM) as a positron emission tomography (PET) ligand for imaging of the metabotropic glutamate receptor subtype 1 (mGluR1) in rat and monkey brains.

Methods

In vivo distribution of [¹⁸F]FITM in brains was evaluated by PET scans with or without the mGluR1-selective antagonist (JNJ16259685). Kinetic parameters of monkey PET data were obtained using the two-tissue compartment model with arterial blood sampling.

Results

In PET studies in rat and monkey brains, the highest uptake of radioactivity was in the cerebellum, followed by moderate uptake in the thalamus, hippocampus and striatum. The lowest uptake of radioactivity was detected in the pons. These uptakes in all brain regions were dramatically decreased by pre-administration of JNJ16259685. In kinetic analysis of monkey PET, the highest volume of distribution (V _T ) was detected in the cerebellum (V _T ?=?11.5).

Conclusion

[¹⁸F]FITM has an excellent profile as a PET ligand for mGluR1 imaging. PET with [¹⁸F]FITM may prove useful for determining the regional distribution and density of mGluR1 and the mGluR1 occupancy of drugs in human brains.     

Synthesis of carbon-11 labelled SCH 39166, a new selective dopamine D-1 receptor ligand,and preliminary PET investigations

SCH 39166 [(—)-trans-6,7,7a,8,9,13b-hexahydro-3-chloro-2-hydroxy-N-methyl-5H-benzo(d)naphtho-(2,1-b)azepine] is a new more selective dopamine D-1 receptor antagonist than the widely used SCH 23390. [¹¹C]SCH 39166 was prepared by N-methylation of the desmethyl compound SCH 40853 [(—)-trans-6,7,7a,8,9,13b-hexahydro-3-chloro-2-hydroxy-5H-benzo(d)naphtho-(2,1-b)azepine] with [¹¹C]methyl iodide. Reaction in acetone with subsequent straight-phase semi-preparative HPLC resulted in 20–30% radiochemical yield (from EOB and decay-corrected) with a total synthesis time of 35–40 min and a radiochemical purity >99%. The specific activity obtained at EOS was about 1500 Ci/mmol (55 GBq/μmol). [¹¹C]SCH 39166 was injected into a Cynomolgus monkey. PET-analysis demonstrated accumulation in the striatum, a region known to have a high density of dopamine D-1 receptors. In a displacement experiment, radioactivity in the striatum was markedly reduced after injection of 6 mg unlabelled SCH 23390, thus demonstrating the specificity and reversibility of [¹¹C]SCH 39166 binding to dopamine D-1 receptors.     

Synthesis of no-carrier-added L- and D-[1-11C]-DOPA

No-carrier-added DL-[1-11C]-DOPA has been synthesized by carboxylation of an alpha-lithioisocyanide with a radiochemical yield of up to 15% without correction for decay. The total synthesis time is 30 min. The resolution of the D- and L-isomers was accomplished within 16 min by HPLC using a chiral stationary phase and a phosphate buffer of pH 4.5 as eluent.     

Synthesis and biological evaluation of [11C]talopram and [11C]talsupram: candidate PET ligands for the norepinephrine transporter

PET and SPECT ligands for the norepinephrine transporter (NET) will be important tools for studying the physiology, pathophysiology and pharmacology of the CNS noradrenergic system in vivo. A series of candidate NET ligands were synthesized and characterized in terms of their affinity for human monoamine transporters. The two most promising compounds, talopram and talsupram, were radiolabeled with carbon-11 and evaluated through biodistribution studies in rats and PET imaging studies in a rhesus monkey. Although both compounds displayed high affinity and selectivity for the human NET in vitro, these compounds did not enter the CNS in adequate amounts to be used in PET imaging studies.     

13C NMR study of the generation of C2- and C3,-deuterated lactic acid by tumoral pancreatic islet cells exposed to D-[1-13C]-, D-[2-13C]- and D-[6-13C]-Glucose in 2H2O

Tumoral pancreatic islet cells of the RIN5mF line were incubated for 120 min in media prepared in ²H₂O and containing D -[1-¹³C]glucose, and D -[2-¹³C]glucose, and D -[6-¹³C]glucose. The generation of C₂- and C₃- deuterated lactic acid was assessed by ¹³C NMR. The interpretation of experimental results suggests that a) the efficiency of deuteration on the C₁ of D-fructose 6-phosphate does not exceed about 47% and 4% in the phosphoglucoisomerase and phosphomannoisomerase reactions, respectively; b) approximately 38% of the molecules of D -glyceraldehyde 3-phosphate generated from D -glucose escape deuteration in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase; and c) about 41% of the molecules of pyruvate generated by glycolysis are immediately converted to lactate, the remaining 59% of pyruvate molecules undergoing first a single or double back-and-forth interconversion with L -alanine. It is proposed that this methodological approach, based on high resolution ¹³C NMR spectroscopy, may provide novel information on the regulation of back-and-forth interconversion of glycolytic intermediates in intact cells as modulated, for instance, by enzyme-to-enzyme tunneling.     

Upregulation of putaminal dopamine D2 receptors in early Parkinson's disease: a comparative PET study with [11C] raclopride and [11C]N-methylspiperone.

Dopamine D2 receptor function was assessed in a PET study with 2 dopamine D2 receptor PET ligands, [11C]raclopride (RAC) and [11C]N-methylspiperone (NMSP), in early Parkinson's disease. METHODS: Seven patients with early Parkinson's disease and 5 healthy volunteers were studied. Each underwent PET both with reversible [11C]RAC and with irreversible [11C]NMSP. RESULTS: Upregulation of dopamine D2 receptors in the putamen contralateral to the predominant symptoms of Parkinson's disease was confirmed using both [11C]RAC and [11C]NMSP. Uptake of [11C]RAC in the contralateral putamen was 105% of uptake in the opposite putamen (P = 0.020). For [11C]NMSP, uptake in the contralateral putamen was 105% of uptake in the ipsilateral putamen (P = 0.011). No significant differences between Parkinson's disease patients and healthy volunteers were detected in any of the studied brain regions using either [11C]RAC or [11C]NMSP. No significant differences between [11C]RAC and [11C]NMSP uptake were detected in the striatum, whereas in the extrastriatal regions, [11C]NMSP showed significantly higher uptake than [11C]RAC both in healthy volunteers and in Parkinson's disease patients. CONCLUSION: This study confirms an increase in dopamine D2 receptors in the putamen contralateral to the predominant symptoms, compared with the ipsilateral putamen, in early Parkinson's disease. This increase was seen both with reversible ligand [11C]RAC and with irreversible ligand [11C]NMSP and thus does not seem a consequence of depleted endogenous dopamine.     

Synthesis and in vivo evaluation of [11C]SA6298 as a PET sigma1 receptor ligand

The potential of a ¹¹C-labeled selective sigma₁ receptor ligand, 1-(3,4-dimethoxyphenethyl)-4-[3-(3,4-dichlorophenyl)propyl]piperazine ([¹¹C]SA6298), was evaluated as a positron emission tomography (PET) ligand for mapping sigma₁ receptors in the central nervous system and peripheral organs. [¹¹C]SA6298 was synthesized by methylation of the desmethyl SA6298 with [¹¹C]CH₃I, with the decay-corrected radiochemical yield of 39 ± 5% based on [¹¹C]CH₃I and with the specific activity of 53 ± 17 TBq/mmol within 20 min from end of bombardment (EOB). In mice, the uptake of [¹¹C]SA6298 was significantly decreased by carrier loading in the brain, liver, spleen, heart, lung, small intestine, and kidney in which sigma receptors are present as well as in the skeletal muscle. Pretreatment with SA6298 also blocked the uptake of [¹¹C]SA6298 by these organs except for the small intestine, but significant displacement of [¹¹C]SA6298 by posttreatment with SA6298 was observed only in the heart, lung, and muscle. In the blocking study with one of the eight sigma receptor ligands, including haloperidol, SA6298, NE-100, (+)-pentazocine, SA4503, (−)-pentazocine, (+)-3-PPP, and (+)-SKF 10,047 (in the order of the affinity for sigma₁ receptor subtype), only SA6298 and an analog SA4503 significantly reduced the brain uptake of [¹¹C]SA6298 to approximately 80% of the control, but the other six ligands did not. Peripherally, the uptake of [¹¹C]SA6298 by the organs described above was decreased predominantly by SA6298 or SA4503, but the blocking effects of the other five ligands except for NE-100 depended on their affinity for sigma₁ receptors. The saturable brain uptake of [¹¹C]SA6298, approximately 20%, was also observed by tissue dissection method in rats and by PET in a cat. Ex vivo autoradiography of the rat brain showed a high uptake in the cortex and thalamus. In the cat brain a relatively high uptake was found in the cortex, thalamus, striatum, and cerebellum. These results have indicated a receptor-mediated uptake of the tracer to some extent in the brain and peripheral organs. However, the tracer has a limited potential for the PET study of the brain receptors because of a relatively high nonspecific binding.     

Difficulties in dopamine transporter radioligand PET analysis: the example of LBT-999 using [18F] and [11C] labelling Part I: PET studies

BackgroundLBT-999 (E)-N-(4-fluorobut-2-enyl)-2β-carbomethoxy-3β-(4′-tolyl)nortropane is a dopamine transporter (DAT) ligand. [¹⁸F]LBT-999 was first labelled with carbon-11; we will now describe its in vivo behaviour in comparison to that of [¹¹C]LBT-999.Methods/ResultsPositron emission tomography (PET) experiments (baboons) confirmed the high affinity/specificity of [¹⁸F]LBT-999 for DAT. The brain regional distribution was in accordance with that of DAT. Pre-treatment with LBT-999 (1 mg/kg iv), but not with desipramine, a norepinephrine (NET) antagonist, reduced the striatum-to-cerebellum ratio by 96%, confirming the specificity for DAT vs. NET. The parent compound decreased rapidly and represented 24.3±5.0% of plasma radioactivity at 30 min pi. Whole-body scans showed an important bone uptake of free fluorine following metabolism of [¹⁸F]LBT-999. In the cerebellum and striatum, distribution volumes increased by 30–40% between 80 and 230 min, suggesting the polluting role of a radiometabolite(s). [¹¹C]LBT-999 exhibited a 40% higher standardized uptake value in the striata. This difference is likely due to N-dealkylation followed by [¹⁸F]fluoride release. 2β-Carbomethoxy-3β-(4′-tolyl) nortropane is then formed, while [¹¹C]2β-carbomethoxy-3β-(4′-tolyl) nortropane is formed following injection of [¹¹C]LBT-999. This metabolite has high affinity for the DAT. In one specific PET experiment, intravenous injection of this metabolite induced a strong displacement of [¹⁸F]LBT-999 in the striata, confirming that this metabolite readily crosses the blood–brain barrier (BBB) and binds to DAT.Conclusions[¹⁸F]LBT-999 is N-dealkylated in vivo to yield (1) a nonradioactive metabolite that crosses the BBB and has a high affinity for the DAT and (2) a [¹⁸F]fluoro-alkyl chain which is further defluorinated. The temporal changes in distribution volumes are consistent with the accumulation of a radiometabolite(s) in the brain. Therefore, the quantification of DAT density with [¹⁸F]LBT-999 is rather difficult.     

Radiosynthesis of a selective dopamine D-1 receptor antagonist: R(+)-7-chloro-8-hydroxy-3-[11C]methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine ([11C]SCH 23390)

Carbon-11 labeled SCH 23390, a selective dopamine D-1 receptor antagonist (Fig. 1b), was prepared by N-alkylation of the nor-methyl precursor with [¹¹C]iodomethane. The product was purified by semi-preparative HPLC and shown to be radiochemically pure at the end of synthesis. The synthesis was completed in approximately 22 min with an average radiochemical yield of 28% (based on [¹¹C]iodomethane) and an average specific activity of approximately 1950 mCi/μmol calculated at the end of synthesis.     

Serial PET studies of human cerebral malignancy with [1-11C]putrescine and [1-11C]2-deoxy-D-glucose

Serial PET measurements of [1-11C]putrescine ([11C]PUT) uptake and glucose metabolic rate (GMR) using [1-11C]2-deoxy-D-glucose ([11C]2DG) were made on eight human subjects with a radiological and, in most cases, pathological diagnosis of primary or metastatic brain tumor. Blood-to-brain influx constants (Ki) were calculated for [11C]PUT. Tumor uptake of 11C after [11C]PUT injection was unidirectional peaking at 15 min. The mean +/- s.d. Kis for [11C]PUT for tumor and normal brain tissue were 0.78 +/- 0.045 and 0.024 +/- 0.007 ml cc-1 min-1, respectively (average of ratio, 3.11) whereas the ratio of GMR for tumor and normal brain tissue was 1.2 +/- 0.5. The mean Ki for four active, high grade astrocytomas was 0.098 +/- 0.030 in contrast to 0.027 +/- 0.008 ml cc-1 min-1 for two patients with low grade astrocytoma. Active high grade astrocytomas also showed marked CT contrast enhancement and regional glucose hypermetabolism. In one subject with brain metastases, both [11C]PUT and GMR correlated with a declining clinical picture in repeated studies over a 4-mo period. PET studies with [11C]PUT provide a better signal:noise ratio than GMR measurements, are useful for locating small glycolytically hypometabolic tumors and, when used in longitudinal studies in a single subject, appear to provide an index of degree of malignancy.